WO2018233589A1 - Procédé destiné à préparer une préparation de lymphocytes t car de qualité clinique par transfection d'adn minicercle dans des lymphocytes t - Google Patents

Procédé destiné à préparer une préparation de lymphocytes t car de qualité clinique par transfection d'adn minicercle dans des lymphocytes t Download PDF

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WO2018233589A1
WO2018233589A1 PCT/CN2018/091772 CN2018091772W WO2018233589A1 WO 2018233589 A1 WO2018233589 A1 WO 2018233589A1 CN 2018091772 W CN2018091772 W CN 2018091772W WO 2018233589 A1 WO2018233589 A1 WO 2018233589A1
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car
target gene
cells
gene
plasmid
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蔡子琪
蔡守锋
韩锦胜
杜萍萍
叶学帅
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蔡子琪
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464493—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a method for preparing clinical grade CAR-T cell preparations by using microcircle DNA transfection technology.
  • Tumor biotherapy has been recognized as the fourth largest means after surgery, radiotherapy, and chemotherapy.
  • Biological therapy mainly includes two types of cellular immunotherapy and antibody-targeted drugs.
  • cell immunotherapy has developed rapidly.
  • immunological detection sites have blocked a large number of new cell preparations such as CTL/TIL, TCR-T, and CART into clinical trials to CD-19/CD20.
  • Targeted CAR-T treatment of B-cell lymphoma as a representative clinical trial obtained more than 90% of the killing efficiency and became a milestone breakthrough. Therefore, CAR-T cell immunotherapy may become an important treatment for tumors.
  • a typical structure of a chimeric antigen receptor includes a scFv linked by a light chain and a heavy chain variable region of a single-chain antibody recognizing an antigen to constitute an extracellular binding region, and a hinge region capable of flexibility of a single-chain antibody. , as well as transmembrane and intracellular signal peptide regions.
  • the extracellular binding region scFv does not need to recognize the antigen in the form of an MHC/antigen peptide complex, but directly recognizes the antigen, and whether the nature of the antigen is a protein or a glycolipid.
  • the size, flexibility and extent of the hinge region determine the ability of the scFv to bind to tumor cell surface antigens.
  • Transmembrane regions play important signaling roles in T cell activation, and are often constructed using transmembrane regions of CD3, CD8, and CD28.
  • the intracellular signal region consists of a costimulatory molecule and a tyrosine activation motif ITAM. Different co-stimulatory molecules differ in their response type, secretion of cytokine levels, and maintenance of cell survival time.
  • the CAR-T prepared by genetic engineering to transfer the CAR structure into T cells has strong specific recognition and killing ability, stable expression and long-lasting function, which is closely related to the construction of CAR structure. CAR's design has undergone three generations of improvement.
  • the first generation CAR only includes CD3 ⁇ or Fc ⁇ RI ⁇ chains; the second generation CAR includes CD3 ⁇ and a costimulatory molecule such as CD28/4-1BB/CD27/ICOS/OX40, these costimulatory molecules enhance CD3 ⁇ signaling; third generation CAR Contains CD3 ⁇ and two costimulatory molecules to make CAR more powerful.
  • a costimulatory molecule such as CD28/4-1BB/CD27/ICOS/OX40, these costimulatory molecules enhance CD3 ⁇ signaling
  • third generation CAR Contains CD3 ⁇ and two costimulatory molecules to make CAR more powerful.
  • other scholars have added one or more vectors encoding CAR and its promoter, and the vector can be further activated by the release of certain cytokines such as IL-12.
  • the signal path of CAR is called the fourth generation CAR.
  • CAR can directly recognize and bind tumor-associated antigen (TAA) on the surface of tumor cells, and transmit antigen signals to T cells, activate T cells and secrete and release large amounts of IL-2, TNF- ⁇ , INF- ⁇ , perforin and Cytokines such as granzymes exert a killing function on tumor cells.
  • TAA tumor-associated antigen
  • a CAR-T cell preparation can be prepared by transfecting the above CAR gene sequence into T cells using a suitable vector.
  • a suitable vector Currently commonly used vectors are mainly two types of viral vectors and non-viral vectors.
  • the viral vector is mainly composed of lentivirus and adenovirus vector.
  • the mechanism of infection of the adenoviral vector system is that the viral DNA cannot be integrated into the chromosome, but only in the nucleus, so the long-term expression of the gene cannot be maintained, and the immune reaction is triggered after repeated application. .
  • the lentiviral vector system can infect the dividing and non-dividing cells, and can integrate the exogenous gene of interest into the chromosome to achieve long-term effective expression of the target gene, and thus is approved by the US FDA for clinical use.
  • the application of viral vectors to prepare clinical grade CAR-T cell preparations still has problems such as low transfection efficiency (40%) and potential safety hazards (gene mutation and tumorigenicity).
  • the foreign gene introduced by the viral vector is randomly integrated into the chromosome, and there is a risk of insertion point mutation and induction of cell transformation or even toxicity and tumorigenesis.
  • Non-viral vectors are commonly used in liposomes, polymers and molecular conjugates.
  • the emerging Sleeping Beauty vector and transposon system are also used for the modification of CART cells, but have low transfection efficiency (20%-40). %), complex process, long time, high cost and other shortcomings.
  • Microcircle DNA is a non-viral vector that has emerged in recent years. It is a small-loop supercoil expression cassette obtained by site-specific recombination by traditional plasmid in E. coli. Studies have used microcircle DNA to carry the target gene and successfully transfected mouse cardiomyocytes and skeletal muscle cells to stably express human vascular epidermal growth factor. Others have successfully transfected human pluripotent stem cells using microcircle DNA. . Studies have shown that non-viral vector microcircle DNA is the best choice for gene therapy, because it lacks resistance marker genes and bacterial replication sequences, significantly improving the safety of microcirculation DNA in clinical application. Whether microcircular DNA can carry the target gene to achieve transfection and modification of human T cells has not been confirmed by experiments.
  • Electrotransfection can destroy some T cell membranes and cause cell death.
  • Our study found that the survival rate of T cells after electrotransfection is about 60% and the growth is slow, but after 3 days of culture, normal expansion and passage can be completely restored, and T The cell phenotype is identical to normal T cells.
  • In vitro and in vivo killing experiments have shown that the prepared target gene-CAR-T cell preparation can identify the corresponding tumor antigen with high efficiency and specificity, and produce high-efficiency and specific tumor killing.
  • the invention Compared with the existing CAR-T preparation technology, the invention has the following outstanding features: (1) successful application of a non-viral microcircle DNA vector for transfection of human T cells; (2) demonstration of a microcircle DNA vector for human T The transfection efficiency is as high as 58%, which is higher than any existing virus or non-viral transfection technology; (3) the expression rate of the target gene-CAR in T cells is as high as 90%, and it is stably expressed with the amplification and passage of T cells; 4)
  • the target gene-CAR-T cell preparation prepared by the invention has strong specific killing function on tumor cells with high expression of the target gene; (5) the target gene-CAR-T cell preparation prepared by the invention does not exist It is clinically safe because of the potential risks of viral replication, bacterial replication, and long-term to tumor.
  • the present invention provides a method for preparing a clinical grade CAR-T cell preparation by microcirculation DNA transfection of T cells, the method comprising the steps of:
  • the steps of the method of the present invention are as follows:
  • the design of the target gene-CAR is as follows: 1. Using the DNAstar8.0 software to analyze and screen the Fab sequence and VH and VL sequences of the anti-target gene for designing the scFv sequence in CAR; 2. Designing the signal peptide The sequence IgGkappa is placed at the front end of the target gene-scFv to direct the CAR structure to penetrate the target cell membrane; 3. The overall framework structure of the target gene-CAR is designed according to the gene sequences of CD8, CD28, CD137 and CD3 ⁇ in Genebank (NCBI). The EcoR1 and BamH1 restriction sites are set at the front and rear ends of the sequence;
  • the synthesis of the target gene-CAR is as follows: 1. The whole gene sequence synthesis of the target gene-CAR is carried out according to the above structure; 2. The synthesized target gene-CAR whole gene sequence is subcloned into the pUC57 vector. Target gene-CAR-pUC57 plasmid;
  • the method is as follows: 1. Applying the plasmid extraction kit to the above-mentioned target gene-CAR-pUC57 plasmid and microcircle DNA vector kit (MC-Easy TM Minicircle DNA Production kit) ;SBI) provided the microcircle DNA parent vector plasmid (pMC.CMV-MCS-EF1-GFP-SV40PolyA) for plasmid extraction to obtain plasmid extract; 2.
  • the target gene obtained in step 1 - CAR-pUC57 and pMC.CMV -MCS-EF1-GFP-SV40PolyA plasmid extract was digested with EcoR1 and BamH1 to obtain a digested product; 3.
  • the target gene obtained in step 2 - CAR-pUC57 and pMC.CMV-MCS-EF1-GFP-SV40PolyA were digested.
  • the product is ligated to obtain a ligation product; 4.
  • the ligation product obtained in step 3 is transformed into a special competent bacterial solution (ZYCY10P3S2TE.coli) provided by the microcircle DNA vector kit, and cultured at 30 ° C, 250 rpm for 90 min.
  • the parental vector plasmid containing the target gene-CAR 50.
  • the 50-200 ⁇ l of the bacterial solution obtained in step 4 was plated on an LB agarose plate containing 50 ⁇ g/ml kanamycin, and cultured overnight at 37 ° C to select a clone. , low-dose extraction of plasmids, digestion, electrophoresis, sequencing ; Such genes were correct, then the step 4 obtained containing the gene of the parent vector plasmid according CAR -80 °C glycerol stock cell suspension, for subsequent extraction -CAR-mcDNA gene of plasmid vector;
  • the target gene-CAR-mcDNA vector plasmid obtained in step (2) is removed from the -80 ° C and inoculated into a Kana containing 50 ⁇ g / ml. 2 ml of LB agarose medium, 30 ° C, 250 rpm, shaking 1-2 h, measuring OD value for conditioning conditions; 2. Inoculating the target gene-CAR parent vector plasmid in 200 ml growth culture Base sterile culture flask, shake at 30 ° C, 250 rpm overnight; 3.
  • Preparation of the target gene-CAR-T cell preparation by the following methods: 1. Isolation of autologous or allogeneic PBMC to obtain T cells, stimulated with 1000 U/ml of IFN- ⁇ , 37 ° C, 5% CO 2 incubator The cells were cultured overnight, and the day 1 day was added with CD3 monoclonal antibody 1 ⁇ g/ml, CD28 monoclonal antibody 1 ⁇ g/ml, and IL-2 500 U/ml, day 2 was added to IL-1510 U/ml, and day 3 was only added with IL-2 500 U/ml. Complete medium culture to expand T cells, change the medium every 2-3 days; 2.
  • the target gene-CAR is designed and synthesized, wherein the designed target gene Fab sequence is selected from the group consisting of: PSCA, PSMA, CEA, EGFR, EGFRvIII, HER2, MSLN, GD2, IL13R ⁇ 2, GPC3, CAIX, L1-CAM, CA125, CD133, FAP, CTAG1B, MUC1, FR- ⁇ , GUCY2C;
  • the CAR sequence designed therein is a third generation CAR sequence containing the scFv-CD3 ⁇ , CD28 and 4-1BB/OX40/ICOS gene sequences, and a second generation CAR sequence containing only the scFv-CD3 ⁇ and CD28 gene sequences, and only contains The first generation CAR sequence of the scFv-CD3 ⁇ gene sequence, and the TRUCK cell CAR sequence containing only the fourth generation; the extraction of the target gene-CAR-mcDNA vector plasmid, wherein the target gene-CAR-mcDNA vector a plasmid, which is a plasmid of any of the genes of interest - CAR-mcDNA;
  • the electroporation T cells prepare a gene-CAR-T cell preparation, wherein the T cells used for transfection are selected from the group consisting of autologous T cells, allogeneic T cells, and each T cell subset.
  • the present invention also encompasses a clinical grade gene-CAR-T cell preparation prepared by the method of the present invention. And the use of the gene of interest-CAR-T cell preparation for the preparation of an antitumor drug.
  • the target gene-CAR containing the EcoR1, BamH1 cleavage site was subcloned into the pUC57 vector plasmid and designated as the target gene-CAR-pUC57 vector plasmid (Fig. 2).
  • Plasmid Extraction The plasmid extraction kit (TIANGEN) respectively above object gene -CAR-pUC57 plasmid and micro circular DNA vector kit (MC-Easy TM Minicircle DNA Production kit; SBI) microring DNA affinity provided by the present vector plasmid (pMC.CMV-MCS-EF1-GFP-SV40PolyA) Plasmid extraction was performed to obtain a plasmid extract.
  • TIANGEN The plasmid extraction kit (TIANGEN) respectively above object gene -CAR-pUC57 plasmid and micro circular DNA vector kit (MC-Easy TM Minicircle DNA Production kit; SBI) microring DNA affinity provided by the present vector plasmid (pMC.CMV-MCS-EF1-GFP-SV40PolyA) Plasmid extraction was performed to obtain a plasmid extract.
  • ligation reaction the above target gene and the vector digested product are subjected to ligation reaction by the following reaction system: target gene digestion product 5 ⁇ l vector digestion product 1 ⁇ l 10 ⁇ T4 DNA ligase buffer 1 ⁇ l T4 DNA ligase 0.6 ⁇ l plus ultrapure water To 10 ⁇ l, the ligation reaction tube was placed at 4 ° C overnight to obtain a ligation product, and the structure is shown in Figure 3.
  • the pH and OD600 values should be adjusted with NaOH and fresh 1 times growth medium to meet the above criteria.
  • PBMC white membrane layer
  • T cell culture and amplification The above T cell suspension was transferred to a suspension culture flask, 1000 U/ml IFN- ⁇ was added, and cultured overnight at 37 ° C in a 5% CO 2 incubator; 1 ⁇ g/ml CD3 was added the next day (day 1). Monoclonal antibody, 1 ⁇ g/ml CD28 mAb, 500 U/ml IL-2 culture; Day2 was added to 10 U/ml IL-15 for 48 hr; Day3 supplemented with 500 U/ml IL-2 complete media continued to amplify passage, every 2-3 Change the day.
  • Electro-transfer configuration of 100 microliter electric rotor: 82 ⁇ l nucleofector solution + 18 ⁇ l supplement, stand at room temperature for use;
  • the target gene-CAR-T cells prepared by electroporation were transferred to a pre-warmed AIM-V serum-free medium containing 500 U/ml IL-2. Increase the number of cells by 10-15 days, change the fluid every 3 days.
  • T cell survival and proliferation activity Electrotransformation damages T cells and has a transient effect on their proliferative activity. Flow cytometry showed that the T cells after electrophoresis for 24 h showed a survival rate of about 60%; the T cell proliferation activity decreased within 3 days after electrotransfection, and the proliferative activity recovered significantly after 3 days.
  • Biotin-protein L binds to the light chain VL and PE-SA in the CAR-T surface single-chain antibody, and then shows the target gene-scFv-CAR.
  • the expression rate on the surface of T cells is as high as 80% or more (Fig. 8).
  • the target gene-CAR protein with a molecular weight of 57 kDa was clearly expressed in CAR-T cells, while the control cells were not expressed (Fig. 9).
  • Preparation of clinical grade target gene-CAR-T cell preparation Take 1-2 ⁇ 10 9 of the target gene-CAR-T cells obtained in 4.3.4, wash the cells by centrifugation 3-4 times in physiological saline, and remove the cell debris according to the conventional preparation. The standard was to transfer the cells into a 200 ml return saline bag/bottle (containing 1% human albumin 2.0 g, IL-2.2 million units) to prepare a clinical grade gene-CAR-T cell preparation.
  • FIG. 1 Schematic diagram of the microcircle DNA parental vector plasmid (pMC.CMV-MCS-EF1-GFP-SV40PolyA).
  • FIG. 1 Schematic representation of the PSCA-CAR-mcDNA vector plasmid extraction product.
  • FIG. 1 Gel electrophoresis of PSCA-CAR-mcDNA vector plasmid.
  • L1 parental vector plasmid (8651 bp);
  • L2 PSCA-CAR-mcDNA vector plasmid (4575 bp).
  • PSCA-CAR-mcDNA vector Plasmid (B) has a transfection efficiency of 58.66% for human T cells, which is significantly higher than that of control plasmid (A).
  • FIG. 8 Flow cytometry at 24 hr after transfection. The expression rate of PSCA-CAR on the surface of T cells was over 80% (Triplicate).
  • FIG. 9 Western blotter detected 57 kDa of CAR protein expression (C), while untransfected T (A) and mock cell (B) expressed only 15 kDa of CD3 ⁇ , but did not express CAR protein.
  • PSCA-CAR-T significantly inhibits AT4 tumor growth with high expression of PSCA.
  • PSCA-CAR gene sequence was subcloned into the pUC57 vector plasmid and designated PSCA-CAR-pUC57 (Fig. 2).
  • Plasmid extraction The microcircular DNA parent vector plasmid (pMC.CMV-MCS-EF1-GFP-SV40PolyA) provided by the above PSCA-CAR-pUC57 plasmid and the microcircle DNA vector kit was respectively applied to the TIANGEN plasmid mini kit. Plasmid extraction was performed to obtain a plasmid extract.
  • Competent bacterial liquid transformation and cloning the ligation product was added to the special competent bacterial liquid (ZYCY10P3S2TE.coli) provided by the microcircle DNA carrier kit, placed in a bacterial incubator, and cultured at 30 ° C, 250 rpm to obtain PSCA- CAR parental vector plasmid bacterial solution; the plasmid bacterial solution was plated on an LB agarose plate containing 50 ⁇ g/ml kanamycin, cultured overnight at 37 ° C, and the bacterial clone was selected the next day; the plasmid was extracted at a small dose, and EcoR1 and BamH1 were double-digested. Electrophoresis, sequencing was correct, and the PSCA-CAR parental vector plasmid was successfully constructed (Fig. 3).
  • the next day take a small amount of medium to measure PH value and OD600 value, adjust the PH value to about 7, OD600 value between 4-6; continue to shake culture for 5-5.5hr ( ⁇ 5.5hr); EcoR1 and BamH1 were digested, gel electrophoresed, and the quality of the PSCA-CAR-mcDNA vector plasmid was tested.
  • the PSCA-CAR-mcDNA vector plasmid was extracted in large quantities according to the QIAGEN EndoFree Plasmid Maxi Kit procedure. EndoFree buffer TE reconstituted the plasmid and quantified.
  • the PSCA-CAR-mcDNA vector plasmid (Fig. 4-5) was stored at -20 °C until use.
  • PSCA-CAR-mcDNA transfection T preparation of PSCA-CAR-T cells 4.
  • PBMC white membrane layer
  • T cell culture and amplification The above T cell suspension was transferred to a suspension culture flask, 1000 U/ml of IFN- ⁇ was added, and cultured overnight at 37 ° C in a 5% CO 2 incubator; the next day was added 1 ⁇ g/ml of CD3 Monoclonal antibody, 1 ⁇ g/ml CD28 monoclonal antibody and 500 U/ml IL-2 culture; Day2 was added to 10 U/ml IL-15 for 48 hr; Day3 was supplemented with 500 U/ml IL-2 complete medium. Change the fluid every 2-3 days.
  • Electrotransfection 82 ⁇ l nucleofector solution was mixed with 18 ⁇ l supplement to prepare the electroporation required for 100 ⁇ l electric rotor; take 5 ⁇ 10 6 of the above T cells, centrifuge at room temperature for 200min for 10min, completely remove the supernatant, and electrolyze 100 ⁇ l Resuspend the cells into a 100 ⁇ l electric rotor; the tip of the gun was placed on the bottom of the cup and gently added to the PSCA-CAR-mcDNA vector plasmid, taking care not to generate bubbles; setting the electrical rotation procedure of the LONZA Nucleofector TM4D electro-rotator, starting the electro-rotation, PSCA-CAR-T The cell preparation is complete.
  • PSCA-CAR-T cells were transferred to the pre-warmed medium, cultured for 10-15 days, and changed every 3 days, and the number of cells was counted periodically.
  • T cell survival rate was about 60% after 24 hours of electroporation, and the damage rate was about 40%.
  • the T cell proliferation activity decreased within 3 days after electroporation, and the proliferative activity recovered significantly after 3 days.
  • PSCA-CAR-T cell preparation Take 1-2 ⁇ 10 9 PSCA-CAR-T cells obtained by amplification in 4.3.3, wash 3-4 times with physiological saline, and transfer 200 ml of normal saline.
  • the bag/bottle (containing 1% human albumin 2.0 g, IL-2.2 million units) was made into a clinical grade PSCA-CAR-T cell preparation.
  • PSCA-CAR-T specifically recognized and killed RTCA cells with positive expression of PSCA, and the killing efficiency was as high as 85.16%, which was significantly higher than that of normal T and mock T in the control group (p ⁇ 0.05; Figure 10). Negative PC-3M cells were almost free of killing.
  • cytokine secretion 24 well plates were added to target cells (RT4 and PC-3M), and cultured for 2 hr at 37 ° C, 5% CO 2 ; normal T, mock T and PSCA were added according to the group. CAR-T effector cells were further cultured for 24 hr; 500 ⁇ l of each group supernatant was centrifuged at 4000 rpm for 10 min to remove particulate matter and polymer, and then the secretion levels of IFN- ⁇ and IL-2 were measured according to the kit instructions.
  • RESULTS PSCA-positive RT4 cells significantly stimulated the secretion of large amounts of IFN- ⁇ and IL-2 by PSCA-CART cells, while normal T and mock T secretions were low (p ⁇ 0.05; Figure 11).
  • NOD/SCID mouse human immune reconstitution taking healthy human PBMC 4 ⁇ 10 7 /0.5ml mouse tail vein injection, feeding for 4 weeks to obtain mouse peripheral blood, flow detection to see CD3 + T lymphocyte expression rate At 67.29%, the expression rate of CD19+ B lymphocytes was 21.5%, and human immune reconstruction was successful (Fig. 12).
  • Humanized NOD/SCID mice model Humanized NOD/SCID mice were injected subcutaneously with 3 ⁇ 10 6 AT4 cells or PC-3M cells, and the tumor formation rate was 100%; The subcutaneous nodules can be seen in the day, 10-15 days to 250-300mm 3 , and the modeling is successful.

Abstract

L'invention concerne un procédé destiné à préparer une préparation de lymphocytes T CAR de qualité clinique, par transfection d'ADN minicercle dans des lymphocytes T. Le procédé consiste à utiliser la technique de transfection efficace d'un vecteur d'ADN minicercle (ADNmc) non viral dans des lymphocytes T afin de préparer une préparation de lymphocytes T CAR de qualité clinique. La technique de transfection d'ADNmc comprend la conception et la synthèse d'un récepteur antigénique chimérique (CAR) portant un gène cible, la construction d'un plasmide vecteur parent du CAR à gène cible, l'extraction d'un plasmide vecteur d'ADNmc du CAR à gène cible, la transfection du plasmide vecteur d'ADNmc du CAR à gène cible dans des lymphocytes T et la préparation de lymphocytes T CAR de qualité clinique, préparée à l'aide de la technique de transfection d'ADNmc. La technique de transfection d'ADNmc présente les caractéristiques d'une efficacité de transfection élevée concernant des lymphocytes T de mammifère, d'une expression stable, d'une fonction stable et de l'absence d'influence sur les propriétés génétiques cellulaires. La préparation de lymphocytes T CAR préparée présente les caractéristiques d'un ciblage fort, d'une efficacité de destruction élevée, d'une sécurité clinique, etc. La technique de transfection d'ADNmc peut être utilisée dans la préparation d'une préparation de lymphocytes T CAR de qualité clinique destinée au traitement de diverses tumeurs solides et de tumeurs du système hématologique et lymphatique.
PCT/CN2018/091772 2017-06-19 2018-06-19 Procédé destiné à préparer une préparation de lymphocytes t car de qualité clinique par transfection d'adn minicercle dans des lymphocytes t WO2018233589A1 (fr)

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CN110129268A (zh) * 2018-02-08 2019-08-16 上海细胞治疗集团有限公司 一种电转后pbmc的培养方法
JP2021520229A (ja) * 2018-03-16 2021-08-19 トーマス ジェファーソン ユニバーシティ 抗ｇｕｃｙ２ｃキメラ抗原受容体の組成物および方法
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JP2021016371A (ja) * 2019-07-23 2021-02-15 株式会社東芝 Ｃａｒ−ｔ細胞の製造方法、核酸導入キャリア及びキット
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